The objective of the proposed research is to identify and characterize small molecule natural product-derived regulators of tumor hypoxia. Solid tumors contain regions under low oxygen tension (hypoxia) and this lack of oxygen contributes to resistance to radiation treatment and chemotherapy. Hypoxia-associated treatment resistance can be caused directly through reduced cellular oxygen concentrations or indirectly through hypoxia-induced modifications in gene expression. Current efforts to overcome hypoxia target the direct effects of hypoxia. We propose a new approach that specifically targets this important indirect effect of hypoxia (alteration of tumor gene expression). The unique focus of this program is to identify and evaluate natural product-derived small molecules that target hypoxia-induced gene expression. A three-pronged approach that combines natural products chemistry with molecular biology will be employed to identify and investigate such small molecules. The first objective will involve isolating and determining the chemical structures of natural product-derived regulators of tumor hypoxia. Such regulators will target a number of processes that are unique to hypoxic tumor cells and key for tumor cell adaptation to hypoxia. A panel of high-throughput bioassays has been established to identify such regulators. Active compounds will be isolated through bioassay-guided fractionation and isolation, and their chemical structures elucidated using a combination of spectroscopic and spectrometric methods. Our group has established the proof of principle for this approach through preliminary research that has identified several potent new hypoxia inducible factor-1 (HIF-1) inhibitors. HIF-1 is a key transcription factor that activates the expression of survival genes under hypoxia. The second objective will identify target genes affected by newly identified active leads. Preliminary studies have revealed that natural product derived small molecules can either selectively inhibit hypoxic activation of HIF-1 or inhibit both hypoxic and chemical activation of HIF-1, depending upon the specific chemical class of each of inhibitor. Inhibition of HIF-1 activation is associated with reduction of the hypoxic induction of HIF-1 target gene vascular endothelial growth factor (VEGF), a potent initiator of tumor angiogenesis. Target genes will be identified by microarray analysis. The third objective will focus on biochemical and cell biological investigations of the mechanisms by which newly identified active natural products regulate hypoxic signaling. Accomplishing these objectives will provide drug leads and molecular probes that target tumor hypoxia and afford new insights into the intracellular pathways affected by these molecules.